New Species and Records of Pleurotheciaceae from Karst Landscapes in Yunnan Province, China

Pleurotheciaceae is a genera-rich and highly diverse family of fungi with a worldwide distribution in aquatic and terrestrial habitats. During the investigation of lignicolous freshwater fungi from karst landscapes in Yunnan Province, China, 15 fresh strains were obtained from submerged decaying wood. Based on the morphology and phylogenetic analysis of a combined LSU, ITS, SSU, and rpb2 sequence dataset, Obliquifusoideum triseptatum, Phaeoisaria obovata, Pleurotheciella brachyspora, Pl. longidenticulata, and Pl. obliqua were introduced as new species, P. synnematica and Rhexoacrodictys melanospora were reported as new habitat records, and P. sedimenticola and Pl. hyalospora were reported as new collections. In addition, based on morphological comparisons and phylogenetic analysis, we accepted Obliquifusoideum into in the family Pleurotheciaceae (Pleurotheciales, Savoryellomycetidae). Freshwater habitats are the primary habitats of Pleurotheciaceae species, and Yunnan Province has the highest concentration and species diversity of Pleurotheciaceae in China.

Obliquifusoideum was established by Dong et al. [6] to accommodate the type species O. guttulatum.The genus is characterized by the following characteristics: superficial, ellipsoidal, black, coriaceous, ostiolate ascomata with a lateral, hyaline-to-dark, subcylindrical

Isolation and Morphological Examination
Fungal colonies on natural substrates were observed using a Guiguang GL-99BI compound stereomicroscope (Guilin Guiguang Instrument Co., Ltd., Guilin, China) and then photographed with a Nikon SMZ1000 stereo zoom microscope (NIKON CORPORATION, Tokyo, Japan).Fungal structures were photographed using a Nikon ECLIPSE Ni-U compound microscope (NIKON CORPORATION, Tokyo, Japan) fitted with a Nikon DS-Ri2 digital camera (NIKON CORPORATION, Tokyo, Japan), as per the guidelines provided by Luo et al. [32] and Senanayake et al. [33].Single spore isolation was conducted by following the methods described by Shen et al. [31].Measurements were made with the Tarosoft (R) Image Frame Work program, and photo plates representing fungal structures were processed in Adobe Photoshop CS5 software (Adobe Systems Inc., San Jose, CA, USA).Herbarium specimens (dry woody branches with fungal material) were deposited in the herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (KUN-HKAS), Kunming, China.The isolates obtained in this study were deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, and the Kunming Institute of Botany Culture Collection Center (KUNCC), Kunming, China.Names of the new taxa were registered in Fungal Names (FN) (https://nmdc.cn/fungalnames/,accessed on 14 June 2024).

Isolation and Morphological Examination
A Trelief TM Hi-Pure Plant Genomic DNA Kit (Beijing TsingKe Biotech Co., Ltd., Beijing, China) was used to extract total genomic DNA from fungal mycelia.DNA amplification was performed by a polymerase chain reaction (PCR).Four partial gene regions, the large subunit of the nuclear ribosomal RNA gene (LSU), the nuclear ribosomal internal transcribed spacer (ITS), the small subunit of the nuclear ribosomal RNA gene (SSU), and the second-largest subunit of RNA polymerase II (rpb2), were used in this study.Sequences of LSU, ITS, SSU, and rpb2 were amplified using primer pairs LR0R/LR5, ITS5/ITS4, NS1/NS4, and fRPB2-5F/fRPB2-7cR, respectively [34][35][36].The amplification was performed in a 25 µL reaction volume containing 9.5 µL of deionized water, a 12.5 µL 2 × Taq PCR Master Mix with blue dye (Sangon Biotech, Shanghai, China), 1 µL of DNA template and 1 µL of each primer (10 µm).The PCR thermal cycling conditions of ITS and SSU were as follows: 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 56 • C for 50 s, elongation at 72 • C for 1 min, and a final extension at 72 • C for 10 min.The LSU thermal cycling conditions were as follows: 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 55 • C for 50 s, elongation at 72 • C for 1 min, and a final extension at 72 • C for 10 min.The rpb2 has a total of 40 cycles, and the conditions are as follows: initial denature at 95 • C for 5 min before entering 40 cycles; then, denaturation occurs at 95 • C for 1 min, annealing at 52 • C for 2 min, extension at 72 • C for 90 s, and finally at 72 • C for 10 min.PCR products were checked on 1% agarose electrophoresis gels stained with Gel Red.The sequencing reactions were carried out with the primers mentioned above by Tsingke Biological Engineering Technology and Services Company, Kunming, China.

Phylogenetic Analyses
BLAST searches were performed to find similar sequences that matched our data.The sequences were aligned using the online multiple alignment program MAFFT version 7 [37], and this alignment was manually optimized in BioEdit v.7.0.5.3 [38].The single-gene dataset was concatenated by SquenceMatrix v.1.7.8 for multi-gene phylogenetic analyses [39].The alignment formats were changed to PHYLIP and NEXUS formats by the AliView and ALigment Transformation EnviRonment (ALTER) website (http://sing.ei.uvigo.es/ALTER/, accessed on 14 June 2024).
Based on phylogenetic analysis, Dong et al. [6] established the genus Obliquifusoideum in Savoryellomycetidae genera incertae sedis.Several phylogenetic studies have yielded the same results, where Obliquifusoideum constitutes an independent lineage that is basal to other genera of Pleurotheciaceae [6,8,9] (Figure 1).We compared the morphology of Obliquifusoideum with genera in Pleurotheciaceae.Obliquifusoideum has semi-immersed-to-superficial, subglobose ascomata, thin peridium, abundant, septate paraphyses, eight-spored, cylindrical asci with an apical ring, and three-septate, fusoid to fusiform ascospores, which are similar to Pleurotheciaceae [1,4,6,12,15].Therefore, we accommodated Obliquifusoideum in Pleurotheciaceae (Pleurotheciales, Savoryellomycetidae). Culture characteristics: Conidia germinate on PDA within 24 h, and germ tubes are produced from both ends.Colonies on PDA after 4 weeks of incubation at room temperature attain a diameter of about 2 cm.Mycelia dry and dense.Colonies on the surface of PDA are raised, with irregular edges, brittle, rough at the surface, and brown-to-dark brown.The reverse is brown-to-dark brown, lighter at the edges, and smooth.

Phaeoisaria synnematica
Material examined: China, Yunnan Province, Qujing City, Luoping County (24 Notes: Phylogenetic analysis showed that our new collections (KUNCC 23-16573 and KUNCC 23-16619) were clustered with the type strains of Phaeoisaria synnematica (NFCCI 4479) (Figure 1).Our new collections are similar to P. synnematica with branched conidiophores, terminal or intercalary, cylindrical, branched conidiogenous cells, and intercalary, lateral-to-terminal, obovoidal or globose-to-subglobose chlamydospores [16].Phaeoisaria synnematica was introduced by Boonmee et al. [16] and collected from the dead bark of Azadirachta indica (Meliaceae) in India, however our two new collections were collected from freshwater habitats.Therefore, we identified our new collections as Phaeoisaria synnematica based on morphological and phylogenetic analysis, and it is the first time to report this species from freshwater habitats in China.
Culture characteristics: Conidia germinate on PDA within 24 h, with germ tubes produced from the apex.Colonies growing on PDA after 3 weeks of incubation at room temperature attain a diameter of about 15 mm.Mycelia dry and dense, with colonies semi-immersed in PDA, with regular edges, a gray, rough surface, and grid texture.The reverse is dark green and smooth.
Fungal Names number: FN 571978.Etymology: Referring to the sexual morph of this fungal has an oblique growing the neck.
Culture characteristics: Ascospores germinate on the PDA within 24 h, with germ tubes produced from the end.Conidia germinate on the PDA within 24 h, with germ tubes produced from both ends.Colonies growing on the PDA after 4 weeks of incubation at room temperature attain a diameter of about 15 mm.Mycelia dry and dense.Colonies ate semi-immersed in PDA, with irregular edges, rough surfaces, protrusions at the center, and celadon.The reverse is dark green and smooth.

Discussion
Phaeoisaria was established by Höhnel [11]: thirty-six epithets are listed in Index Fungorum [20], and only one species is known as a sexual morph.The asexual morph of Phaeoisaria is characterized by compactly adpressed synnematous conidiophores, which are subcylindrical, curved conidiogenous cells with multiple denticulate conidiogenous loci, and ellipsoidal-to-obovoid or clavate, aseptate, hyaline conidia; the asexual morph exhibited little morphological differences between the species in the genus [13,16,50].
Therefore, species of this genus are distinguished primarily by molecular evidence and supplemented by the sizes of synnemata, conidiogenous cells, and conidia [9,13,53].
In this study, we observed the reproduced asexual morph of Phaeoisaria synnematica on the PDA medium, and we also observed produced chlamydospores of this species but did not find the reduced single conidiogenous cell arising from aerial hyphae conidiophores and septate conidia, which were mentioned by Boonmee et al. [16].In addition, five species, including P. annesophieae, P. fasciculata, P. goiasensis, P. guttulate, and P. loranthacearum, also observed some morphologies in the culture that differed from other species of this genus, including producing chlamydospores, a lack of synnemata, either reduced or short, arising from aerial hyphae conidiophores, and septate conidia [1,9,17,54,55].However, these characteristics were not observed in all species on the surface of the natural substrates [12,16,49,56,57].Therefore, the difference in growth substrates has a great influence on the morphogenesis of species in this genus, and we should pay more attention to the colony on culture to provide as many morphological characteristics of the same species as possible and provide more evidence to identify the species of Phaeoisaria.
We compiled fifty-seven freshwater Pleurotheciaceae species belonging to eleven genera, as shown in Table 2, and these species were collected from different climatic zones around the world.These results indicate that the species of this family have strong adaptability to different climates, and freshwater habitats are their main living environment (Figure 1).Asia is the region with the highest number of reported species, especially in Yunnan Province, China, where more than half (29/57, 50.88%) of these species are found in Yunnan Province.The suitable, complex, and changeable climate and geographical environment of Yunnan Province provide corresponding growth conditions for different variations in organisms.This is true not only for species of Pleurotheciaceae but also for other fungal taxa [29,30,58].

Figure 1 .
Figure 1.RAxML tree based on combined LSU, ITS, SSU, and rpb2 sequence data of Pleurotheciaceae (Pleurotheciales) and Savoryellaceae (Savoryellales).Bootstrap support values for maximum likelihood (ML) greater than 75% and Bayesian posterior probabilities (PP) greater than 0.95 are given as ML/PP above the nodes.Newly obtained sequences are indicated in red, and ex-type strains are in bold.

Figure 1 .
Figure 1.RAxML tree based on combined LSU, ITS, SSU, and rpb2 sequence data of Pleurotheciaceae (Pleurotheciales) and Savoryellaceae (Savoryellales).Bootstrap support values for maximum likelihood (ML) greater than 75% and Bayesian posterior probabilities (PP) greater than 0.95 are given as ML/PP above the nodes.Newly obtained sequences are indicated in red, and ex-type strains are in bold.

Table 2 .
Species of Pleurotheciaceae from freshwater habitats.